Evaluation of tumorigenic potential of high yielding cloned MDCK cells for live-attenuated influenza vaccine using in vitro growth characteristics,metastatic gene expression and in vivo nude mice model

Several mammalian cell lines, including Madin–Darby canine kidney (MDCK) cells have been approved by regulators for manufacturing of human vaccines. A new MDCK 9B9-1E4 cloned cell line has been created which is capable of producing live attenuated influenza vaccine (LAIV) with high yield. This cell line was shown to be non tumorigenic in eight week old adult athymic nude mouse model. This property is desirable for vaccine production and is unique to this cell line and is not known to be shared by other MDCK cell lines that are currently used for vaccine production. This significant difference in tumorigenic phenotype required further characterization of this cell line to ensure its safety for use in vaccine production. This is particularly important for LAIV production where it is not possible to incorporate a virus inactivation and/or removal step during manufacturing. Characterization of this cell line included extensive adventitious agent testing, tumorigenicity and oncogenicity assessment studies. Here, we describe the development of tumorigenic MDCK cell lines for use as positive controls and in vitro methods to aid in the evaluation of the tumorigenicity of MDCK 9B9-1E4 cloned cells. Tumorigenic MDCK cells were successfully generated following Hras and cMyc oncogene transfection of MDCK 9B9-1E4 cloned cells. In this study we demonstrate the lack of tumorigenic potential of the MDCK 9B9-1E4 cloned cell line in adult athymic nude mice model.     

Both mucosal and systemic routes of immunization with the live,attenuated NYVAC/simian immunodeficiency virus SIV(gpe) recombinant vaccine result in gag-specific CD8(+) T-cell responses in mucosal tissues of macaques

As most human immunodeficiency virus (HIV) infection occurs via mucosal surfaces, an important goal of vaccination may be the induction of virus-specific immune responses at mucosal sites to contain viral infection early on. Here we designed a study in macaques carrying the major histocompatibility complex class I Mamu-A(*)01 molecule to assess the capacity of the highly attenuated poxvirus NYVAC/simian immunodeficiency virus (SIV) SIV(gpe) vaccine candidate administered by the intranasal, intramuscular, or intrarectal route to induce mucosal immunity. All macaques, including one naive macaque, were exposed to SIV(mac251) by the intrarectal route and sacrificed 48 h after infection. The kinetics of immune response at various time points following immunization with NYVAC/SIV(gpe) and the anamnestic response to SIV(mac251) at 48 h after challenge were assessed in blood, in serial rectal and vaginal biopsy samples, and in tissues at euthanasia with an SIV(mac) Gag-specific tetramer. In addition, at euthanasia, antigen-specific cells producing gamma interferon or tumor necrosis factor alpha from the jejunum lamina propria were quantified in all macaques. Surprisingly, antigen-specific CD8(+) T cells were found in the mucosal tissues of all immunized macaques regardless of whether the vaccine was administered by a mucosal route (intranasal or intrarectal) or systemically. In addition, following mucosal SIV(mac251) challenge, antigen-specific responses were mainly confined to mucosal tissues, again regardless of the route of immunization. We conclude that immunization with a live vector vaccine results in the appearance of CD8(+) T-cell responses at mucosal sites even when the vaccine is delivered by nonmucosal routes.     

Intranasal vaccine from whole Leishmania donovani antigens provides protection and induces specific immune response against visceral leishmaniasis

Visceral leishmaniasis is a protozoan disease associated with high fatality rate in developing countries. Although the drug pipeline is constantly improving, available treatments are costly and live-threatening side effects are not uncommon. Moreover, an approved vaccine against human leishmaniasis does not exist yet. Using whole antigens from Leishmania donovani promastigotes (LdAg), we investigated the protective potential of a novel adjuvant-free vaccine strategy. Immunization of mice with LdAg via the intradermal or the intranasal route prior to infection decreases the parasitic burden in primary affected internal organs, including the liver, spleen, and bone marrow. Interestingly, the intranasal route is more efficient than the intradermal route, leading to better parasite clearance and remarkable induction of adaptive immune cells, notably the helper and cytotoxic T cells. In vitro restimulation experiments with Leishmania antigens led to significant IFN-γ secretion by splenocytes; therefore, exemplifying specificity of the adaptive immune response. To improve mucosal delivery and the immunogenic aspects of our vaccine strategy, we used polysaccharide-based nanoparticles (NP) that carry the antigens. The NP-LdAg formulation is remarkably taken up by dendritic cells and induces their maturation in vitro, as revealed by the increased expression of CD80, CD86 and MHC II. Intranasal immunization with NP-LdAg does not improve the parasite clearance in our experimental timeline; however, it does increase the percentage of effector and memory T helper cells in the spleen, suggesting a potential induction of long-term memory. Altogether, this study provides a simple and cost-effective vaccine strategy against visceral leishmaniasis based on LdAg administration via the intranasal route, which could be applicable to other parasitic diseases.     

Research progress in human infection with avian influenza H7N9 virus

Since the identification of the novel reassortant avian influenza A (H7N9) virus in China in 2013, until Jun 30, 2017, the virus has caused five epidemic waves leading to a total of 1,552 human infections, with a fatality rate of about 40%. In the spring of 2017, highly pathogenic avian influenza (HPAI) H7N9 virus emerged and has caused 25 human infections. The HPAI H7N9 virus has some biological differences from the LPAI one, such as its multiple basic amino acid residues on HA leading to its independence on trypsin for replication. The pathogenicity of the HPAI H7N9 virus to experimental animals or humans is still unclear. A(H7N9) vaccine development for pandemic preparedness is ongoing, including the reassortment (H7N9/PR8) reverse genetic based vaccine, the virus like particle (VLP) vaccine, the intranasal live attenuated influenza vaccine (LAIV), the non-adjuvant Vero cell culture-derived inactivated whole-virus vaccine, the MDCK culture-derived vaccine, the H7 DNA vaccine and the recombinant replicative H7N9 virus (H7N9-53TM) vaccine. Five neuramidinase resistant sites of A(H7N9) virus isolated from patients have been reported. Some alternative drugs have been studied, such as DAS181 (Fludase), ribavirin, troglitazone and minocycline. Persistent surveillance and enhanced global control are essential to fight against human infections with A(H7N9) virus.     

Mucosal Immunization with Integrase-Defective Lentiviral Vectors Protects against Influenza Virus Challenge in Mice

Recent reports highlight the potential for integrase-defective lentiviral vectors (IDLV) to be developed as vaccines due to their ability to elicit cell-mediated and humoral immune responses after intramuscular administration. Differently from their integrase-competent counterpart, whose utility for vaccine development is limited by the potential for insertional mutagenesis, IDLV possess a mutation in their integrase gene that prevents genomic integration. Instead, they are maintained as episomal DNA circles that retain the ability to stably express functional proteins. Despite their favorable profile, it is unknown whether IDLV elicit immune responses after intranasal administration, a route that could be advantageous in the case of infection with a respiratory agent. Using influenza as a model, we constructed IDLV expressing the influenza virus nucleoprotein (IDLV-NP), and tested their ability to generate NP-specific immune responses and protect from challenge in vivo. We found that administration of IDLV-NP elicited NP-specific T cell and antibody responses in BALB/c mice. Importantly, IDLV-NP was protective against homologous and heterosubtypic influenza virus challenge only when given by the intranasal route. This is the first report demonstrating that IDLV can induce protective immunity after intranasal administration, and suggests that IDLV may represent a promising vaccine platform against infectious agents.     

Development and evaluation of a DNA vaccine based on Helicobacter pylori urease B: failure to prevent experimental infection in the mouse model

BACKGROUND: The development of a vaccine against Helicobacter pylori has become a priority to prevent major morbidity and mortality associated with this infection. Our goal was to prepare and evaluate a DNA vaccine based on the urease B gene (ureB). METHODS: The ureB gene of H. pylori was amplified and cloned into the eukaryotic expression vector pcDNA3.1/TOPO. Plasmid DNA was purified from transformed Escherichia coli cells and used to immunize mice by the intragastric, intramuscular, intrarectal (40 micro g each) and intranasal (16 micro g) route, three doses every 2 weeks, with CpG oligodeoxynucleotide (ODN) as adjuvant. Four weeks after the third dose, animals were orally challenged with Helicobacter felis and were sacrificed 6 weeks later. The stomach was stained to detect the presence of infection. RESULTS: Despite in vitro confirmation of successful cloning and functionality of the ureB gene with expression of a protein morphologically and antigenically identical to urease B, the DNA vaccine did not perform well in vivo. Immunization of mice produced a weak immune response. Overall, intrarectal and intranasal administration seemed more immunogenic than other routes. Protection against challenge was modest and nonsignificant, and slightly better on animals immunized by the intramuscular and intranasal route. CONCLUSION: A DNA vaccine based on H. pylori urease B was poorly immunogenic and nonprotective at the conditions evaluated. Higher doses, better adjuvants or a prime-boost approach may circumvent these limitations.     

Removing residual DNA from Vero-cell culture-derived human rabies vaccine by using nuclease

The clearance of host cell DNA is a critical indicator for Vero-cell culture-derived rabies vaccine. In this study, we evaluated the clearance of DNA in Vero-cell culture-derived rabies vaccine by purification process utilizing ultrafiltration, nuclease digestion, and gel filtration chromatography. The results showed that the bioprocess of using nuclease decreased residual DNA. Dot-blot hybridization analysis showed that the residual host cell DNA was <100 pg/ml in the final product. The residual nuclease in rabies vaccine was less than 0.1 ng/ml protein. The residual nuclease could not paly the biologically active role of digestion of DNA. Experiments of stability showed that the freeze-drying rabies virus vaccine was stable and titers were >5.0 IU/ml. Immunogenicity test and protection experiments indicated mice were greatly induced generation of neutralizing antibodies and invoked protective effects immunized with intraperitoneal injections of the rabies vaccine. These results demonstrated that the residual DNA was removed from virus particles and nuclease was removed by gel filtration chromatography. The date indicated that technology was an efficient method to produce rabies vaccine for human use by using nuclease.     

Production of cell culture (MDCK) derived live attenuated influenza vaccine (LAIV) in a fully disposable platform process

The majority of influenza vaccines are manufactured using embryonated hens' eggs. The potential occurrence of a pandemic outbreak of avian influenza might reduce or even eliminate the supply of eggs, leaving the human population at risk. Also, the egg‐based production technology is intrinsically cumbersome and not easily scalable to provide a rapid worldwide supply of vaccine. In this communication, the production of a cell culture (Madin‐Darby canine kidney (MDCK)) derived live attenuated influenza vaccine (LAIV) in a fully disposable platform process using a novel Single Use Bioreactor (SUB) is presented. The cell culture and virus infection was maintained in a disposable stirred tank reactor with PID control of pH, DO, agitation, and temperature, similar to traditional glass or stainless steel bioreactors. The application of this technology was tested using MDCK cells grown on microcarriers in proprietary serum free medium and infection with 2006/2007 seasonal LAIV strains at 25–30 L scale. The MDCK cell growth was optimal at the agitation rate of 100 rpm. Optimization of this parameter allowed the cells to grow at a rate similar to that achieved in the conventional 3 L glass stirred tank bioreactors. Influenza vaccine virus strains, A/New Caledonia/20/99 (H1N1 strain), A/Wisconsin/67/05 (H3N2 strain), and B/Malaysia/2506/04 (B strain) were all successfully produced in SUB with peak virus titers ≥8.6 log₁₀ FFU/mL. This result demonstrated that more than 1 million doses of vaccine can be produced through one single run of a small bioreactor at the scale of 30 L and thus provided an alternative to the current vaccine production platform with fast turn‐around and low upfront facility investment, features that are particularly useful for emerging and developing countries and clinical trial material production. Biotechnol. Bioeng. 2010;106: 906–917. © 2010 Wiley Periodicals, Inc.     

M cell DNA vaccination for CTL immunity to HIV

To facilitate invasion, reovirus has evolved to attach to M cells, a specialized epithelium residing within the follicle-associated epithelium that covers mucosal inductive tissues. Thus, we questioned adapting reovirus protein sigma1 to ferry DNA vaccines to the mucosa to immunize against HIV. Three expression plasmids encoding HIV(Ba-L) gp160, cytoplasmic gp140, and secreted gp140 were tested in mice as protein sigma1-poly-L-lysine-DNA complexes (formulated vaccine) via the intranasal route. Evaluation of cell-mediated immunity showed that the formulated gp160 DNA vaccine was more effective for stimulating envelope (Env)-specific CTL responses in lungs, lower respiratory lymph nodes (LN), cervical LN, submaxillary gland LN, and spleens. Three doses of vaccine were required for CTL responses, and intranasal naked DNA immunizations were ineffective. The greatest CTL activity was observed between weeks 8 and 10 for gp160-vaccinated mice, and activity remained detectable by week 16. These Env-specific CTL responses were perforin dependent in peripheral tissues, but mostly Fas dependent in the lungs. These Env-specific CTLs also produced IFN-gamma. Mice vaccinated with the formulated gp160 DNA vaccine showed potent antiviral immunity against vaccinia virus-env replication in ovaries. Thus, compared with live vectors, protein sigma1-mediated DNA delivery represents an alternative mucosal formulation for inducing cellular immunity against HIV-1.     

Intranasal immunization with a lipooligosaccharide-based conjugate vaccine from nontypeable Haemophilus influenzae enhances bacterial clearance in mouse nasopharynx

Nontypeable Haemophilus influenzae (NTHi) is a major cause of otitis media in children. We investigated whether intranasal immunization with a detoxified lipooligosaccharide-tetanus toxoid (dLOS-TT) conjugate vaccine would generate protective immunity against NTHi in a mouse model of nasopharyngeal clearance. The results demonstrated that intranasal immunization with dLOS-TT plus adjuvant cholera toxin (CT) significantly induced LOS-specific IgA antibodies in mouse external secretions, especially in nasal wash (90-fold), bronchoalveolar lavage fluid (25-fold), saliva (13-fold) and fecal extract (three-fold). LOS-specific IgA antibody-forming cells were also found in mucosal and lymphoid tissues with their highest numbers in the nasal passage (528 per 10(6) cells). In addition, the intranasal immunization elicited a significant rise in LOS-specific IgG (32-fold) and IgA (13-fold) in serum. For the immunized mice which had been challenged through the nose with 10(7) live NTHi strain 9274 cells, the vaccine group showed a significant reduction (74-77%) of NTHi, compared to that of control groups with CT alone or dLOS plus CT (P<0.05). Negative correlations were found between bacterial counts and the levels of nasal wash IgA or IgG, saliva IgA and serum IgG. The clearance of five heterologous strains was investigated and revealed a significant clearance of strains 3198, 5657 and 7502 but not of strains 1479 and 2019. These data suggest that intranasal immunization with dLOS-TT vaccine elicits both mucosal and systemic immunity against NTHi and enhances bacterial clearance from nasopharynx in mice. Such a vaccine and vaccination regime may be applicable to humans with an appropriate formulation.     

Effectiveness of topically administered neutralizing antibodies in experimental immunotherapy of respiratory syncytial virus infection in cotton rats.

Initial studies of the prophylactic effect of parenterally administered respiratory syncytial virus (RSV)-neutralizing antibodies in cotton rats indicated that virus replication in lung tissues was restricted when animals with preexisting antibody titers in serum of 1:100 or more (as measured by plaque reduction) were challenged intranasally with 10(4) PFU of virus. Subsequently, a therapeutic effect of parenterally administered RSV antibodies (present in human gamma globulin) was demonstrated in both cotton rats and owl monkeys. Parenteral inoculation of RSV-infected cotton rats or owl monkeys with purified human immunoglobulin licensed for intravenous administration in humans (IVIG) effected a 10(-1.7) to 10(-2.7) reduction in the level of pulmonary virus at the height of infection. Because of these encouraging results, we examined topical administration of IVIG to determine whether it was also effective and whether it offered an advantage over the parenteral route with regard to simplicity and the dose required for full therapeutic effect. IVIG (0.025 g/kg) administered topically by the intranasal route to anesthetized cotton rats at the height of RSV infection effected a 10(2.2)-fold reduction in viral titers of pulmonary tissues and a complete clearance of detectable virus in 92% of the animals within 24 h. In contrast, 4 g of IVIG per kg was required to produce a comparable therapeutic effect when the material was administered parenterally. Thus, the therapeutic effect of IVIG was 160 times greater by the topical route than by parenteral inoculation.     

Pharmacokinetic analysis of polyamide nucleic-acid-cell penetrating peptide conjugates targeted against HIV-1 transactivation response element

We have demonstrated that polyamide nucleic acids complementary to the transactivation response (TAR) element of HIV-1 LTR inhibit HIV-1 production when transfected in HIV-1 infected cells. We have further shown that anti-TAR PNA (PNA(TAR)) conjugated with cell-penetrating peptide (CPP) is rapidly taken up by cells and exhibits strong antiviral and anti-HIV-1 virucidal activities. Here, we pharmacokinetically analyzed (125)I-labeled PNA(TAR) conjugated with two CPPs: a 16-mer penetratin derived from antennapedia and a 13-mer Tat peptide derived from HIV-1 Tat. We administered the (125)I-labeled PNA(TAR)-CPP conjugates to male Balb/C mice through intraperitoneal or gavage routes. The naked (125)I-labeled PNA(TAR) was used as a control. Following a single administration of the labeled compounds, their distribution and retention in various organs were monitored at various time points. Regardless of the administration route, a significant accumulation of each PNA(TAR)-CPP conjugate was found in different mouse organs and tissues. The clearance profile of the accumulated radioactivity from different organs displayed a biphasic exponential pathway whereby part of the radioactivity cleared rapidly, but a significant portion of it was slowly released over a prolonged period. The kinetics of clearance of individual PNA(TAR)-CPP conjugates slightly varied in different organs, while the overall biphasic clearance pattern remained unaltered regardless of the administration route. Surprisingly, unconjugated naked PNA(TAR) displayed a similar distribution and clearance profile in most organs studied although extent of its uptake was lower than the PNA(TAR)-CPP conjugates.     

Live Attenuated Influenza Vaccine Provides Superior Protection from Heterologous Infection in Pigs with Maternal Antibodies without Inducing Vaccine-Associated Enhanced Respiratory Disease

Control of swine influenza A virus (IAV) in the United States is hindered because inactivated vaccines do not provide robust cross-protection against the multiple antigenic variants cocirculating in the field. Vaccine efficacy can be limited further for vaccines administered to young pigs that possess maternally derived immunity. We previously demonstrated that a recombinant A/sw/Texas/4199-2/1998 (TX98) (H3N2) virus expressing a truncated NS1 protein is attenuated in swine and has potential for use as an intranasal live attenuated influenza virus (LAIV) vaccine. In the present study, we compared 1 dose of intranasal LAIV with 2 intramuscular doses of TX98 whole inactivated virus (WIV) with adjuvant in weanling pigs with and without TX98-specific maternally derived antibodies (MDA). Pigs were subsequently challenged with wild-type homologous TX98 H3N2 virus or with an antigenic variant, A/sw/Colorado/23619/1999 (CO99) (H3N2). In the absence of MDA, both vaccines protected against homologous TX98 and heterologous CO99 shedding, although the LAIV elicited lower hemagglutination inhibition (HI) antibody titers in serum. The efficacy of both vaccines was reduced by the presence of MDA; however, WIV vaccination of MDA-positive pigs led to dramatically enhanced pneumonia following heterologous challenge, a phenomenon known as vaccine-associated enhanced respiratory disease (VAERD). A single dose of LAIV administered to MDA-positive pigs still provided partial protection from CO99 and may be a safer vaccine for young pigs under field conditions, where dams are routinely vaccinated and diverse IAV strains are in circulation. These results have implications not only for pigs but also for other influenza virus host species.     

Induction of genital immunity by DNA priming and intranasal booster immunization with a replication-defective adenoviral recombinant.

Mice immunized through different routes such as i.m., intradermally, or intratracheally with a DNA vaccine to rabies virus developed high titers of serum Ab but only borderline levels of mucosal Abs determined from vaginal secretions. DNA vaccines given by either route enhanced vaginal IgA and IgG2a secretion upon a subsequent intranasal booster immunization with an E1-deleted adenoviral recombinant expressing the same Ag of rabies virus. DNA vaccine priming reduced the Ab response to the adenoviral Ags and counterbalanced the impaired B cell response to the rabies virus Ag expressed by the adenoviral recombinant in mice preimmune to adenovirus. The vaginal B cell response could further be enhanced by using the Th2-type cytokines IL-4 or IL-5 as genetic adjuvants concomitantly with the DNA vaccine before intranasal booster immunization with the recombinant vaccine.     

Comparison of the intravenous and intraperitoneal routes of administration of tritiated thymidine in studies of cell production in the gastrointestinal tract of the rat

Incorporation of 3H-Thymidine into DNA-synthesizing cells of the gastrointestinal tract of the rat was examined following administration of the isotope by intraperitoneal and intravenous routes. Estimates of whole tissue incorporation expressed as DPM/mg dry weight and of proliferating cells expressed as DPM/crypt or gland in the different segments of the gut indicated no differences in the degree of 3H-TdR uptake into DNA following intraperitoneal or intravenous routes of administration. The possibility of misdirected I.P. injections was examined following injection of 3H-TdR into the cecum or bladder. DPM/mg wet weight of gastrointestinal tissues indicated reduction in the uptake of 3H-TdR into DNA of intestinal tissues following intracecal and intrabladder administration of 3H-Tdr. The intraperitoneal route of administration of 3H-TdR appears to be equally effective in the distribution of the isotope into different segments of the gut when compared to the intravenous route and is a more convenient method in studies of cell production in the gastrointestinal tract of the rat.     

Enhancement of the Immunogenicity and Protective Efficacy of a Mucosal Influenza Subunit Vaccine by the Saponin Adjuvant GPI-0100

Identification of safe and effective adjuvants remains an urgent need for the development of inactivated influenza vaccines for mucosal administration. Here, we used a murine challenge model to evaluate the adjuvant activity of GPI-0100, a saponin-derived adjuvant, on influenza subunit vaccine administered via the intranasal or the intrapulmonary route. Balb/c mice were immunized with 1 µg A/PR/8 (H1N1) subunit antigen alone or in combination with varying doses of GPI-0100. The addition of GPI-0100 was required for induction of mucosal and systemic antibody responses to intranasally administered influenza vaccine and significantly enhanced the immunogenicity of vaccine administered via the intrapulmonary route. Remarkably, GPI-0100-adjuvanted influenza vaccine given at a low dose of 2×1 µg either in the nares or directly into the lungs provided complete protection against homologous influenza virus infection.     

Mycobacterium tuberculosis Infection Interferes with HIV Vaccination in Mice

Tuberculosis (TB) has emerged as the most prominent bacterial disease found in human immunodeficiency virus (HIV)-positive individuals worldwide. Due to high prevalence of asymptomatic Mycobacterium tuberculosis (Mtb) infections, the future HIV vaccine in areas highly endemic for TB will often be administrated to individuals with an ongoing Mtb infection. The impact of concurrent Mtb infection on the immunogenicity of a HIV vaccine candidate, MultiHIV DNA/protein, was investigated in mice. We found that, depending on the vaccination route, mice infected with Mtb before the administration of the HIV vaccine showed impairment in both the magnitude and the quality of antibody and T cell responses to the vaccine components p24Gag and gp160Env. Mice infected with Mtb prior to intranasal HIV vaccination exhibited reduced p24Gag-specific serum IgG and IgA, and suppressed gp160Env-specific serum IgG as compared to respective titers in uninfected HIV-vaccinated controls. Importantly, in Mtb-infected mice that were HIV-vaccinated by the intramuscular route the virus neutralizing activity in serum was significantly decreased, relative to uninfected counterparts. In addition mice concurrently infected with Mtb had fewer p24Gag-specific IFN-γ-expressing T cells and multifunctional T cells in their spleens. These results suggest that Mtb infection might interfere with the outcome of prospective HIV vaccination in humans.     

Evaluation of the sublingual route for administration of influenza H5N1 virosomes in combination with the bacterial second messenger c-di-GMP

Avian influenza A H5N1 is a virus with pandemic potential. Mucosal vaccines are attractive as they have the potential to block viruses at the site of entry, thereby preventing both disease and further transmission. The intranasal route is safe for the administration of seasonal live-attenuated influenza vaccines, but may be less suitable for administration of pandemic vaccines. Research into novel mucosal routes is therefore needed. In this study, a murine model was used to compare sublingual administration with intranasal and intramuscular administration of influenza H5N1 virosomes (2 μg haemagglutinin; HA) in combination with the mucosal adjuvant (3',5')-cyclic dimeric guanylic acid (c-di-GMP). We found that sublingual immunisation effectively induced local and systemic H5N1-specific humoral and cellular immune responses but that the magnitude of response was lower than after intranasal administration. However, both the mucosal routes were superior to intramuscular immunisation for induction of local humoral and systemic cellular immune responses including high frequencies of splenic H5N1-specific multifunctional (IL-2+TNF-α+) CD4+ T cells. The c-di-GMP adjuvanted vaccine elicited systemic haemagglutination inhibition (HI) antibody responses (geometric mean titres ≥ 40) both when administered sublingually, intranasally and inramuscularly. In addition, salivary HI antibodies were elicited by mucosal, but not intramuscular vaccination. We conclude that the sublingual route is an attractive alternative for administration of pandemic influenza vaccines.     

Cellular immune responses in children and adults receiving inactivated or live attenuated influenza vaccines

The patterns of cellular immune responses induced by live attenuated influenza vaccine (LAIV) versus those of the trivalent inactivated influenza vaccine (TIV) have not been studied extensively, especially in children. The goals of this study were to evaluate the effects of TIV and LAIV immunization on cellular immunity to live influenza A virus in children and adults and to explore factors associated with variations in responses to influenza vaccines among individuals. A gamma interferon (IFN-gamma) flow cytometry assay was used to measure IFN-gamma-producing (IFN-gamma+) NK and T cells in peripheral blood mononuclear cell cultures stimulated with a live influenza A virus strain before and after LAIV or TIV immunization of children and adults. The mean percentages of influenza A virus-specific IFN-gamma+ CD4 and CD8 T cells increased significantly after LAIV, but not TIV, immunization in children aged 5 to 9 years. No increases in the mean levels of influenza A virus-reactive IFN-gamma+ T cells and NK cells were observed in adults given LAIV or TIV. TIV induced a significant increase in influenza A virus-reactive T cells in 6-month- to 4-year-old children; LAIV was not evaluated in this age group. The postvaccination changes (n-fold) in the percentages of influenza A virus-reactive IFN-gamma+ T and NK cells in adults were highly variable and correlated inversely with the prevaccination percentages, in particular with that of the CD56(dim) NK cell subset. In conclusion, our findings identify age, type of vaccine, and prevaccination levels of immune reactivity to influenza A virus as factors significantly associated with the magnitude of cellular immune responses to influenza vaccines.     

Treatment of malaria in a mouse model by intranasal drug administration

The goal of this work was to investigate intranasal dihydroartemisinin (DHA) delivery as a non-invasive method for treatment of malaria. ICR female mice were infected with Plasmodium berghei ANKA, a model for severe malaria with similarities to the human disease. DHA, at a dose of 2 × 5 mg/kg/day, was administered to mice either intranasally or i.p. Two dosage regimens were tested: prophylaxis and treatment. Parasitemia was monitored every other day, from the time of infection, by thin smears prepared from tail blood. The survival rates in prophylaxis and treatment regimens were 93% and 75%, respectively, for intranasal DHA and this route was at least as effective as the i.p. route used for comparison. All mice in the untreated control and placebo groups succumbed due to the parasitemia. The results show that DHA nasal administration to mice was highly efficient in the treatment of Plasmodium infection in infected rodents. This novel mode of drug administration may be considered as an alternative to conventional treatment.